In order to obtain a natural-type protein produced by animal cells through culturing the animal cells or in order to produce a desired protein through culturing animal cells to which a gene encoding the desired protein has been introduced, the culture medium usually contains an extract from mammals for supporting proliferation of these animal cells, in addition to basic nutrients, such as salts, saccharides, amino acids, and vitamins. Specifically, serum such as fetal bovine serum is added to a culture medium in an amount of 5 to 20%. Such serum derived from a mammal accounts for 75 to 95% of the total cost of the medium, and has a disadvantage that stable proliferation is not achieved due to a lot-to-lot variation in quality. In addition, a mammal-derived serum a cannot be sterilized by, for example, an autoclave; thus, it is likely of undergoing contamination with viruses or mycoplasma. Even though many of them are harmless, the contamination may be an additional unknown factor from the viewpoint of stable production.
Recently, it has been concerned about alleged involvement of extracts from mammals with, for example, mad cow disease, bovine spongiform encephalopathy (BSE), transmissible spongiform encephalopathy (TSE), or Creutzfeld-Jakob disease (CJD), and there is a demand for culture media not containing extracts from mammals for culturing animal cells, from the viewpoint of safety. In addition, serum contains 500 or more different proteins, resulting in complication of isolation and purification of a desired protein produced by the cells, from the culture medium.
In order to solve the above-described problems, serum-free culture media have been developed for culturing animal cells in the absence of serum. In methods for serum-free culture, serum-free culture solutions have been developed that contains plasma proteins such as fetuin, transferrin, and albumin; hormones such as steroid hormones and insulin; growth factors; and nutritional factors such as amino acids and vitamins, as alternatives to serum.
Proteins used in serum-free culture, such as fetuin, insulin, transferrin, and a growth factor, are purified from serum or are produced as recombinant proteins from recombinant organisms. The use of these proteins still has disadvantages such as, for example, inclusion of substances derived from living organisms, even in trace amounts, use of expensive materials, and a lot-to-lot variation in culture.
Recently, serum-free culture using protein hydrolysates has also been developed, but this also has the same disadvantages, i.e., inclusion of substances derived from living organisms, high cost, and a lot-to-lot variation in the products obtained. Thus, the methods are not satisfactory for production of useful proteins.
Accordingly, there is a demand for a culture method using a synthetic culture medium that contains the minimum amount of substances derived from living organisms, is inexpensive, is with a small lot-to-lot variation, and can enhance production of a large amount of protein. Recently, analysis of behavior of glucose and amino acids in culture solutions in fed-batch culture has revealed that addition of an increased amount of glutamic acid to the fed-batch culture contributes to high throughput production of antithrombin (NPL 1). Unfortunately, these findings are based on only studies on CHO cells expressing glutamine synthetase, and validation has not been conducted with normal CHO cells. Furthermore, there is no finding on individual amino acids other than glutamic acid. In addition, the throughput of protein production is still insufficient. Accordingly, there is a high demand for culture using a synthetic culture medium that further increases the throughput.
Since the fed-batch culture involves supply of nutrients that have been consumed by cells proliferating and surviving in the culture medium while the culture medium is generally not drawn out from a batch until the completion of the culture, the osmotic pressure of the culture medium gradually increases mainly due to an acid or base added for the pH adjustment. An excessive increase in osmotic pressure is harmful for cells. Saccharides and amino acids serving as nutrients in the culture medium and by-products generated by metabolism also cause an increase in osmotic pressure.
Also reported is a method for increasing a desired product by addition of a specific peptide to the culture medium (NPL 2).